PROJECT SUMMARY Gut microbial colonization in early postnatal life plays a crucial role in mammalian intestinal development. It has long been postulated that DNA methylation, as an epigenetic mechanism to control gene expression, is involved in intestinal host-microbial interactions. The high turnover of intestinal epithelial cells throughout life makes intestinal stem cells (ISCs) critically important for gut function. Remarkably, however, how the mechanisms underlying the effects of the gut microbiota on DNA methylation to regulate the emergence and behavior of adult ISCs remain poorly understood. Improving our understanding of this process is fundamental for human health because intestinal epithelium is one of the major tissue targets of inflammation and tumorigenesis, and aberrant DNA methylation as well as alterations of the gut microbiota are increasingly recognized to be critical for disease pathogenesis. The proposed research extends our previous work that identified a subset of 3? CpG islands (3? CGIs) that are methylated in ISCs during the suckling period in mice. We demonstrated that 3? CGI methylation transmits an epigenetic memory associated with stable gene activation in adult ISCs. In addition, we discovered that 3? CGI methylation is uniquely vulnerable to gut microbiota perturbations. Therefore, the goal of the proposed research is to further elucidate the mechanisms of epigenetic regulation in developing ISCs. Our hypothesis is that postnatally established epigenetic memory by 3? CGI methylation provides a developmental pathway for regulating intestinal host-microbiome interactions with lifelong functional consequences. We propose the following three specific aims: (i) Define the mechanism by which 3? CGI methylation regulates intestinal gene activation, (ii) Define the mechanism by which the gut microbiota regulates 3? CGI methylation, and (iii) Define the long-term function of microbiota-responsive 3? CGI methylation. We will capitalize on recent technological advancements enabling isolation of Lgr5+ ISCs; apply state-of-the-art techniques to achieve the ultimate genome-wide, unbiased assessment of the microbiome effects on the ISC epigenome; and use cutting-edge organoid and CRISPR epigenome editing tools to dissect the microbiota-mediated epigenetic mechanisms that regulate ISC function. The successful completion of these studies will yield important insights into the functional role of DNA methylation during intestinal development, advancing our understanding of the molecular basis of gene-environment interactions in the intestine. Furthermore, the mechanistic insight gained from these studies offers great promise for development of interventions and treatments of intestinal diseases.